p21CIP is a potent cell cycle inhibitor often up-regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21CIP. However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21CIP in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21CIP to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over-expression studies showed that p21CIP is required to induce differentiation. However, the expression of p21CIP needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21CIP caused early down-regulation of mitotic Cyclins and up-regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over-activation of MYC did not release the proliferative block imposed by p21CIP and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21CIP takes part in a mitosis control driving MYC-induced cellular growth into differentiation. J. Cell. Physiol. 227: 2069–2078, 2012. © 2011 Wiley Periodicals, Inc.